Little is known of the molecular basis of smooth-muscle cell development in the microvessels of the adult lung in pulmonary hypertension (PH). Using quantitative and immunogold electron microscopy techniques we report the development of microvascular precursor smooth-muscle cells (PSMCs) expressing alpha-smooth-muscle actin (alphaSMA), a first marker of smooth-muscle cell differentiation, in rats with hyperoxic PH. Increase in the frequency of distal (alveolar wall) vessels with alphaSMA cells preceded (Pchi2 < 0.02, Day 4) the increase in proximal (alveolar duct) vessels (Pchi2 < 0.02, Day 14). The smallest vessel with cells expressing alphaSMA (< 50 micrometer in diameter) increased most with time (Pchi2 < 0.001). Immunopositive PSMCs were rare in normal lung and frequent in hyperoxia. Well-developed filament arrays decorated with alphaSMA were detected in intermediate cells early in hyperoxia (Day 4). Similar filament networks were detected later in fibroblasts recruited to vessel walls (Days 7 to 14). By Day 28, cells derived from fibroblasts formed several layers in the vessel wall and expressed dense alphaSMA filament arrays, in either a central domain or mesh. Thus, intermediate cells are the source of cells expressing alphaSMA early in the microvessels in hyperoxic pulmonary hypertension and fibroblasts of cells in the late stage-the time of intense neomuscularization of the microvessels.